Scientific context of the project
The use of antibiotics in intensive farming contributes to the emergence of bacteria resistant to these molecules, limiting the effectiveness of treatments available in both veterinary and human medicine. The phenomena of antibiotic resistance observed in recent years are, in this context, a major concern for human and animal public health and have led to an awareness of the need for a more reasoned and coordinated use of available antibiotics. Among the fish farming sectors, particular attention must be paid to fish farming, which represents a major potential source of spread of resistant bacteria, via water but also via fish intended for consumption.
Unlike land-based farms (pigs, poultry, etc.), there is no monitoring plan for the fish farming sector to assess the prevalence of resistant bacteria at the farm level or in filleting or processing plants. The only French data currently available from the field are the results of antibiotic susceptibility tests on isolates of clinical origin received by Resapath. These data are nevertheless few and remain patchy.
However, the French fish farming industry is still regularly exposed to antibiotics, despite the efforts made by professionals in recent years to reduce their use. While 60% of the sites surveyed were users in 1997, this proportion was reduced to about one site in two in 2007 (Pisciculture census, SSP 2007). It should be noted that the low number of molecules that can be used in fish farming is also problematic.
Therefore, no truly operational monitoring tool is available to establish a relevant overview of antimicrobial resistance in the fish farming industry, and there is no possibility to establish trends in the evolution of antimicrobial resistance due to the lack of a reference point. It remains complex, therefore, to assess the impact of changes in zootechnical and prophylactic practices in this sector, such as the intensification of vaccination against red mouth disease (or yersiniosis) due to Yersinia ruckeri since the 2000s, or to be able to detect an "emergence" of resistant strains as has been done for third-generation cephalosporins for terrestrial sectors.
In freshwater fish, and rainbow trout in particular (Oncorhynchus mykiss), Yersinia ruckeri and Aeromonas salmonicida are, together with Flavobacteriumpsychrophyllum, the main agents responsible for bacterial diseases. These represent a significant economic cost for the industry.
Yersinia ruckeri, Gram- Enterobacterium, is the agent of the so-called red mouth disease, mainly in trout. Vaccination with commercial vaccines helps to effectively prevent the disease. Antibiotic therapy is based on the use of quinolone antibiotics such as flumequine, which has an MA for trout. This disease has a significant economic cost for the industry and is characterized by successive seasonal relapses. Studies on the antibiotic sensitivity of clinical and/or environmental isolates of this potentially pathogenic species are few and although there is an American reference (CLSI) for the performance of antibiotic susceptibility tests and the determination of MICs, there is not yet a defined clinical threshold..
Aeromonas salmonicida sbsp salmonicida is the agent of furunculosis, an almost pandemic disease since only Australia is free of it. A serious pathology of freshwater fish and also representing a significant economic cost for the industry, the only existing treatment is curative with the use of antibiotics of the tetracycline family (oxytetracycline) and/or quinolones (oxolinic acid, flumequine). Although the antibiotic susceptibility of Aeromonas salmonicida strains has been the subject of numerous studies, the collections described are made up of strains isolated over a limited period of time and territory. The Aeromonas genus is particularly interesting to study because within this genus, species potentially pathogenic to fish (A. salmonicida and A. hydrophila to a lesser extent) and species potentially pathogenic to humans (A. hydrophila, A. caviae, A. sobria...) are found side by side. Moreover, this genus is indigenous to the aquatic environment and plasmid gene transfers between Aeromonas and E. coli have been demonstrated. Aeromonas can also harbour resistance genes to critical antibiotics (fluoroquinolones, cephalosporins).
1 - Constitution of a strain list by mutualisation of the partners' collections
A collection of isolates of Yersinia ruckeri and Aeromonas spp. (more than 159 strains of Yersinia sp. and 169 strains of Aeromonas sp. available) will be built up by bringing together strains isolated or collected by the different partners over the last 40 years. Additional strains may be collected from departmental laboratories carrying out routine analyses in fish bacteriology. For each isolate, all available information (commemorative, registration forms) will be collected, recorded and analysed.
2. Culture, identification and characterization of strain antibiotic resistance profiles
Each of the isolates in this collection, some of which have been preserved for decades, will be re-cultured to verify its purity and DNA extraction will be performed. All purified isolates will be re-identified using a technique that takes into account recent taxonomic developments for these two genera and avoids the need for identification based solely on phenotypic criteria, which is always problematic for environmental strains.
The study of the sensitivity of each isolate to a selection of antibiotics, including those used in the past or currently in aquaculture (trimethoprim-sulfonamide, oxolinic acid, flumequine, chloramphenicol (banned since 1995), oxytetracycline and forfenicol), as well as families of medical or epidemiological interest (beta-lactam antibiotics, etc.), will be carried out by determining MICs and the antibiotic susceptibility test according to the methods described by the CLSI. It will thus be possible to assess the concordance between the two methods and to attempt to establish critical epidemiological thresholds that will allow better categorization of strains in routine.
The parallel use of the two methods also aims to develop on the one hand an expertise of the methods in the laboratories and on the other hand an in-depth knowledge of the strains and their level of resistance.
The detection of a selection of resistance genes such as qnr, tet, sul, blaTEM can be done by PCR.
Concerning Yersinia ruckeri isolates, a biotyping will be carried out to describe the emergence of biotype 2 (non-mobile), which is increasingly encountered in the field and is the cause of vaccine failures due to its evolution compared to the commercial vaccine strain. It will be of particular interest to observe the period of appearance of this biotype in western France.
3. Creation of a common strain library
All the purified strains shared in this study by the partners will be stored in a strain library located at LabOcéa. For each strain, the identification profiles obtained will be associated, as well as the antibiotic resistance profiles and the associated MICs. The strains will be made available to the project partners.
This work will make it possible to :
- establish a regional reference collection of pathogenic Yersinia ruckeri and Aeromonas spp. strains isolated from fish from the 1970s to the present day. This strain library can then be completed with other isolates and other species (establishment of collaboration agreements).,
- have initial elements of comparison between MICs and antibiotic susceptibility tests, with the aim of having a finer methodology for evaluating antibiotic resistance that could eventually be transferred to departmental laboratories ;
- carry out a report on the temporal evolution over nearly 30 years of antimicrobial resistance for two of the main bacterial species involved in recurrent pathologies in freshwater fish in fish farms,
- obtain data on the prevalence of resistance genes in the collected isolates, which will make it possible to better understand and evaluate the potential for gene transfer and thus the role of reservoir and vector of resistance genes in farmed fish.
This work, which has the advantage of initiating a regional dynamic at the level of the "Antibiotic resistance" theme for the fish farming sector, will lay the methodological bases that will allow the implementation of relevant monitoring tools, within the framework of the Resapath, whose data will be used to assess the effectiveness of measures to reduce the use of antibiotics..